Project Summary Engagement of IgG Fc receptors (Fc?Rs) on natural killer cells, monocytes, macrophages, or dendritic cells may play a critical role in preventing lentivirus infection. For example, a broadly neutralizing monoclonal antibody (IgG1 b12) provides far more complete protection of macaques against vaginal challenge with SHIV when Fc?R engagement and antibody-dependent cell-mediated virus inhibition (ADCVI) are allowed to occur. Moreover, recombinant gp120 (rgp120) immunization of humans elicits an ADCVI antibody response whose magnitude correlates with reduced rates of sexually transmitted HIV infection. Thus, it is likely that an effective HIV vaccine will depend not only on interactions between antibody and antigen but also on interactions between Fc?Rs and their Fc ligands. We will investigate two biological properties of antibody, IgG subclass and Fc glycosylation, that impact its ability to bind to Fc?Rs and consequently to inhibit HIV-1. Since IgG2 binds poorly to most Fc?Rs and decreased sialic acid or fucose content increases Fc-Fc?R affinity, we will test the following hypotheses: 1) the IgG2 response to rgp120 vaccine varies between individuals, and the ADCVI activity elicited by rgp120 vaccine is inversely proportional to gp120-specific IgG2 levels;2) anti-gp120 antibodies elicited by rgp120 immunization vary with respect to Fc glycosylation pattern, and Fc glycosylation is a determinant of ADCVI activity;and 3) modifications in the sialic acid and fucose content of IgG1 b12 will increase the potency and breadth of neutralizing and ADCVI activity. Our long-term goal is to construct vaccines eliciting antibodies that optimally engage Fc?Rs. We will accomplish the following specific aims: 1) Measure gp120-specific IgG subclasses elicited by vaccination with rgp120, and determine the association between subclass and ADCVI activity. Using sera from vaccinated subjects, gp120- specific IgG subclasses will be quantified by ELISA. Subclass distribution will be correlated with ADCVI responses measured previously;2) Quantify the sialic acid and fucose content in IgG Fc elicited by rgp120, and determine the association between Fc glycosylation and anti-HIV-1 activity. Lectin ELISA and other methods will be applied to determine Fc glycosylation patterns[unreadable]particularly the frequency of fucosylated and sialylated glycans[unreadable]of gp120 affinity-purified IgG;and 3) Isolate low sialic acid and low fucose variants of the broadly reactive mAb IgG1 b12, and determine the affect of Fc glycosylation changes on anti-HIV-1 activity. Fucose and sialic acid content will be modified and neutralizing and ADCVI functions measured. It is likely that vaccine responses could be purposefully biased toward a specific distribution of subclasses or Fc glycoforms. Given the obstacles to developing broadly reactive, vaccine-induced antibodies, efforts at improving antibody function by altering Fc represents a promising, and perhaps essential, complementary approach to HIV vaccine development. Relevance We propose studying certain properties of antibody that might be crucial for preventing HIV infection. Such studies may lead to methods of improving the ability of antibody to inhibit HIV and eventually to the development of effective HIV/AIDS vaccines.